The present invention is directed to a method for producing a new nematocidal composition particularly useful against plant parasitic nematodes and also a process to prevent damage resulting from nematode infestation. The method for production of the composition involves heating a pH-adjusted fermentation broth of microorganisms to a temperature of at least about 100xc2x0 C. for at least about 15 minutes.
Plant parasitic nematodes cause serious economic damage to many agricultural crops around the world. The nematodes in this group are microscopic worms and are, in general, obligate parasites of plants. They feed mostly on the roots of host plants; however, several genera are known to parasitize above-ground parts including stems, leaves and flowers as well. Almost all the plant species of economic importance are susceptible to infection by some species of nematodes (notable exceptions are in the marigolds and asparagus). For example, root knot nematodes (RKN), (Meloidogyne spp.) are capable of parasitizing more than 3,000 species of crop plants. These plants include agronomic crops, vegetables, fruits, flowering trees and shrubs. Nematodes reportedly cause crop loss equivalent to more than six billion dollars in the United States alone and more than one hundred billion dollars around the world.
The symptoms due to parasitic nematode injury vary widely depending on the plant host, the nematode species, age of the plant, geographical location and climatic and external environmental conditions. In general, an overall patchy appearance of plants in a field is considered indicative of nematode infestation. More specifically, nematode injury results in galling of the roots (abnormal swelling in the tissue due to rapid multiplication of cells in the cortical region) caused by species of root knot (Meloidogyne spp.) and cyst (Heterodera spp.) nematodes, lesions (localized, discolored areas) caused by lesion nematodes (Pratylenchus spp.), suppression of cell division resulting in stubby roots (Trichodorus spp.), growth abnormalities including crinkling or twisting of above-ground parts (Aphelenchoides spp.), and even cell necrosis (death) in some cases. Plant parasitic nematodes may be endoparasitic in nature, as in the case of the root-knot and lesion nematodes, or ectoparasitic as in the dagger nematode (Xiphinema spp.) and lance nematode (Hoplolaimus spp.). Nematodes can be vectors of plant viruses and are also known to induce disease complexes predisposing plants to infection by other plant pathogenic fungi and bacteria.
Chemical nematocides, either soil fumigants or non-fumigants, have been in use for many years and are among the few feasible options for countering nematodes. At present, the process involves repeated applications of synthetic chemicals to the ground prior to planting the crop. These chemicals are extremely toxic to organisms besides nematodes and many of them may pose serious threats to the environment. With the renewed emphasis on clean water and air by environmental groups and governmental agencies, and the detection of many of these active ingredients or the metabolites thereof in ground water and several non-target organisms, there has been serious concern as to the manufacture and/or use of these chemicals. One of the most effective, economical, and widely used nematocides, DBCP (1,2-dibromo-3-chloropropane), found in ground water has been judged to induce male sterility and possible carcinogenesis. Another widely used chemical, EDB (ethylene dibromide), has also been found in ground water.
Yet another very common insecticide-nematocide, aldicarb (2-methyl-2-(methylthio)-propionaldehyde-O-(methylcarbamoyl)oxime), has been found to be acutely toxic. Aldicarb has been found in ground water in several regions of United States. Carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl methylcarbamate) and 1,3-D (1,3-dichlorpropane), two very commonly used nematocides, are under special review by EPA because of their avian toxicity and possible carcinogenic effects. More recently, the decision by EPA to limit and eventually discontinue the use of the soil fumigant, methyl bromide, for agricultural purposes presents a threat to the efficiency and quality of agricultural production in the United States.
Natural isolates such as N-acetyl-D-glucosamine, which may be derived from microorganisms which are the waste products of industrial fermentation processes, have been disclosed as nematocidal in U.S. Pat. No. 5,057,141.
Biopesticides have been developed as an alternative to chemical pesticides. They are obtained by fermentation and can be used either as crude biomass or purified. Typically, fermentations are carried out at temperatures in the range of 20-40xc2x0 C. For example, submerged fermentation at 28-30xc2x0 C. of Paecilomyces fumosoroues fungal isolate ATCC No. 20874 produces fungal biomass for control of nematode infestation as disclosed in U.S. Pat. No. 5,360,607; whole fermentation broth from fermentation at 28xc2x0 C. of Streptomyces thermoarchaensis NCIB 12015 is disclosed as nematocidal in U.S. Pat. No. 5,182,207; fermentation broth obtained from fermentation of Streptomyces cyaneogriseus noncyanogenus NRRL 15773 at 28xc2x0 C. is effective against nematodes as disclosed in U.S. Pat. No. 5,439,934; and fermentation broth obtained by fermentation of the fungus Myrothecium verrucaria at temperatures of from 25 to 30xc2x0 C. is disclosed as nematocidal in U.S. Pat. No. 5,051,255.
Heating of an infested biomass, as disclosed in U.S. Pat. No. 4,229,442, at a temperature of at least 125xc2x0 C. with water in an amount ranging from 1-5 times the dry weight of the biomass, may combat nematodes.
However, there is still a need for the development of new and effective nematocides. It is therefore an object of this invention to provide a method for the production of nematocidal compositions which are effective and inexpensive.
The invention is directed to a method of producing a nematocide from fermentation broths of microorganisms by heating a fermentation broth to a temperature of at least about 100xc2x0 C. after adjusting the pH to about pH 2 or below, or about pH 8 or above. The nematocide thus produced can be used to prevent plant damage and/or limit the growth of nematodes.
The present invention is directed to a method for improving biopesticidal activity of materials produced by fermentation comprising the steps of:
fermenting a bacterium or fungus to obtain a fermentation broth;
adjusting the pH of said fermentation broth to a pH of below about 2.5 with a biologically acceptable acid, or to a pH of above about 8 with a biologically acceptable alkali or base; and
heat treating said fermentation broth to a temperature of at least about 100xc2x0 C. for at least about 15 minutes, then cooling to ambient temperature to obtain a pH-adjusted, heat treated composition having improved biopesticidal activity;
with the proviso that said fungus is not Myrothecium verrucaria when the pH of said fermentation broth is adjusted to a pH of below about 2.5.
The invention is also directed to a composition comprising
a heat-treated, pH adjusted fermentation broth of a fungus or bacterium selected from the group consisting of Gibberella fujikuroi, Streptomyces erythraeus, Bacillus sphaericus, Bacillus thuringiensis and Fusarium moniliforme; 
wherein said broth is pH adjusted either to a pH of below about 2.5 with a biologically acceptable acid, or to a pH of above about 8 with a biologically acceptable alkali or base;
and wherein said broth is heated to a temperature of at least about 100xc2x0 C. for at least about 15 minutes, then cooled to ambient temperature.
The invention is also directed to a method for control of nematodes on plants which comprises the step of administering to the locus, soil or seed of plants in need of such treatment, a nematocidally effective amount of a composition formed by heating a pH adjusted fermentation broth from a fungus or bacterium;
wherein said broth is adjusted to a pH of below about 2.5 with a biologically acceptable acid, or to a pH of above about 8 with a biologically acceptable alkali or base;
and wherein said broth is heated to at least 100xc2x0 C. for at least 15 minutes;
with the proviso that said fungus is not Myrothecium verrucaria when the pH of said fermentation broth is adjusted to a pH of below about 2.5.
The composition may be administered at a rate of from about 1 to about 200 pounds per acre.
The invention is also directed to a method for producing a nematocidal composition comprising the steps of:
a) fermenting a bacterium or fungus to obtain a fermentation broth;
b) suspending said broth in an aqueous solution;
c) adjusting the pH of said broth in aqueous solution to a pH below about pH 2.5 with a biologically acceptable acid;
d) heating the pH-treated broth of step c) to a temperature of at least about 100xc2x0 C. for at least about 15 minutes, then cooling to ambient temperature; and,
e) recovering said composition;
with the proviso that said fungus is not Myrothecium verrucaria. 
Preferably, the pH is adjusted below about 2; and most preferably, the pH is adjusted below about 1.5.
The pH of the composition formed in step d) may be adjusted to a range of about pH 4 to about pH 8. The fermentation broth of step a) may have water-soluble biomass and water-insoluble biomass; and the water-soluble biomass may be separated from said water-insoluble biomass, and the water-insoluble biomass may be suspended in aqueous solution in step b).
The invention is also directed to a method for producing a nematocidal composition comprising the steps of:
a) fermenting a bacterium or fungus to obtain a fermentation broth;
b) suspending said broth in an aqueous solution;
c) adjusting the pH of said broth in aqueous solution to a pH above about pH 8 with a biologically acceptable alkali or base;
d) heating the pH-treated broth of step c) to a temperature of at least about 100xc2x0 C. for at least about 15 minutes, then cooling to ambient temperature;
e) recovering said composition.
Preferably, the pH is adjusted above about 9 and most preferably the pH is adjusted above about 10.
The pH of the composition formed in step d) may be adjusted to a range of about pH 4 to about pH 8. The fermentation broth of step a) may have water-soluble biomass and water-insoluble biomass; and the water-soluble biomass may be separated from said water-insoluble biomass, and the water-insoluble biomass may be suspended in aqueous solution in step b).
For the practice of any aspect of this invention, at least one compound such as herbicides, antimicrobials, fungicides, insecticides, plant growth regulators or nutrients may be added to the heat-treated, pH adjusted compositions.